Invasive Aspergillosis (IA) is a devastating and frequently fatal infection in immunocompromised patients, in part because of poor early detection tools. It presently kills 52% of those infected. The ability to promptly launch antifungal therapy for Aspergillosis and other invasive fungal diseases is critical for positive patient outcomes. However, early detection is complicated by low levels of antigenic markers that signal disease onset and by the poor specificity of current diagnostic assays. The aim of this proposal is to develop an antibody- based diagnostics platform comprised of new monoclonal antibodies coupled with the ultrasensitive detection capabilities of surface enhanced Raman spectroscopy and gold nanoparticle sandwich assays. To address the challenges in the early detection of IA, a collaborative research team of scientists from the University of Utah and North Dakota State University has been assembled and includes immunologists with expertise in monoclonal antibody development and screening for Aspergillus spp; medical microbiologists with strengths in basic mycology, and in fungal assay development and validation; analytical chemists and nanotechnologists with a strong focus in the creation of novel, ultrasensitive and selective diagnostic tests; and infectious disease clinicians with proficiency in the design and implementation of patient-oriented research. This R21 grant application will advance this concept using Aspergillus fumigatus (A. fumigatus) - the most common cause of IA - as the proof-of-principle target. The specific aims for this R21 grant application are: (1) To generate unique, stage-specific biomarkers and associated monoclonal antibodies (mAbs) for A. fumigatus. Murine mAbs will be generated against cellular components and secreted products of A. fumigatus. (2) To identify and optimize multiplexed mAb combinations for a sandwich immunoassay based on surface enhanced Raman scattering (SERS), and mAb-coated gold nanoparticle labels. (3) To assess the performance of the assay using biological sample matrices. The detection strategy created through the successful completion of this project will redefine the diagnosis of IA through augmented sensitivity and specificity, permitting treatment at the earliest stages of disease. PUBLIC HEALTH RELEVANCE: Invasive aspergillosis (IA) is a devastating, frequently fatal infection in immunocompromised and immunosup- pressed patients. Unfortunately, the ability to promptly launch antifungal therapy for IA and other invasive fun- gal diseases is compromised by the poor clinically sensitivity, clinical specificity, and prognostic value of exist- ing diagnostic tools. Until IA can be reliably identified at early stages, the morbidity/mortality attendant with this dreadful disease will remain unacceptably high. The overall goal of this project is to develop a rapid, cost effec- tive laboratory test that will serve as a cornerstone for the confident early diagnosis of IA.